Emmanuelle Jeannette

Protein Tyrosine Kinases and Protein Tyrosine Phosphatases Are Involved in Abscisic Acid-Dependent Processes in Arabidopsis Seeds and Suspension Cells

Protein tyrosine (Tyr) phosphorylation plays a central role in many signaling pathways leading to cell growth and differentiation in animals. Tyr phosphorylated proteins have been detected in higher plants, and the roles of protein Tyr phosphatases and protein Tyr kinases in some physiological responses have been shown. We investigated the involvement of Tyr phosphorylation events in abscisic acid (ABA) signaling using a pharmacological approach. Phenylarsine oxide, a specific inhibitor of protein Tyr phosphatase activity, abolished the ABA-dependent accumulation of RAB18 (responsive to ABA 18) transcripts. Protein Tyr kinase inhibitors like genistein, tyrphostin A23, and erbstatin blocked the RAB18 expression induced by ABA in Arabidopsis (Arabidopsis thaliana). Stomatal closure induced by ABA was also inhibited by phenylarsine oxide and genistein. We studied the changes in the Tyr phosphorylation levels of proteins in Arabidopsis seeds after ABA treatment. Proteins were separated by two-dimensional gel electrophoresis, and those phosphorylated on Tyr residues were detected using an anti-phosphotyrosine antibody by western blot. Changes were detected in the Tyr phosphorylation levels of 19 proteins after ABA treatment. Genistein inhibited the ABA-dependent Tyr phosphorylation of proteins. The 19 proteins were analyzed by matrix-assisted laser-desorption ionization time-of-flight/time-of-flight mass spectrometry. Among the proteins identified were storage proteins like cruciferins, enzymes involved in the mobilization of lipid reserves like aconitase, enolase, aldolase, and a lipoprotein, and enzymes necessary for seedling development like the large subunit of Rubisco. Additionally, the identification of three putative signaling proteins, a peptidyl-prolyl isomerase, an RNA-binding protein, and a small ubiquitin-like modifier-conjugating enzyme, enlightens how Tyr phosphorylation might regulate ABA transduction pathways in plants.

Plasmalemma Abscisic Acid Perception Leads to RAB18 Expression via Phospholipase D Activation in Arabidopsis Suspension Cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K+ channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca2+influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression ofRAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.

Induction of abscisic acid-regulated gene expression by diacylglycerol pyrophosphate involves Ca2+ and anion currents in arabidopsis suspension cells

Diacylglycerol pyrophosphate (DGPP) was recently shown to be a possible intermediate in abscisic acid (ABA) signaling. In this study, reverse transcription-PCR of ABA up-regulated genes was used to evaluate the ability of DGPP to trigger gene expression in Arabidopsis (Arabidopsis thaliana) suspension cells. At5g06760, LTI30, RD29A, and RAB18 were stimulated by ABA and also specifically expressed in DGPP-treated cells. Use of the Ca2+ channel blockers fluspirilene and pimozide and the Ca2+ chelator EGTA showed that Ca2+ was required for ABA induction of DGPP formation. In addition, Ca2+ participated in DGPP induction of gene expression via stimulation of anion currents. Hence, a sequence of Ca2+, DGPP, and anion currents, constituting a core of early ABA-signaling events necessary for gene expression, is proposed.

Arabidopsis thaliana lipid phosphate phosphatase 2 is involved in abscisic acid signalling in leaves

Lipid phosphate phosphatases (LPPs, E.C. 3.1.3.4) catalyse the dephosphorylation of diacylglycerol pyrophosphate (DGPP) and phosphatidic acid (PA), which are secondary messengers in abscisic acid (ABA) signalling. In this study, we investigated the effect of ABA on the expression of AtLPP genes as they encode putative ABA-signalling partners. We observed that AtLPP2 expression was down-regulated by ABA and we performed experiments on Atlpp2-2, an AtLPP2 knockout mutant, to determine whether AtLPP2 was involved in ABA signalling. We observed that Atlpp2-2 plantlets contained about twice as much PA as the wild-type Col-0 and exhibited higher PA kinase (PAK) activity than Col-0 plants. In addition, we showed that ABA stimulated diacylglycerol kinase (DGK) activity independently of AtLPP2 activity but that the ABA-stimulation of PAK activity recorded in Col-0 was dependent on AtLPP2. In order to evaluate the involvement of AtLPP2 activity in guard cell function, we measured the ABA sensitivity of Atlpp2-2 stomata. The inhibition of stomatal opening was less sensitive to ABA in Atlpp2-2 than in Col-0. Watered and water-stressed plants of the two genotypes accumulated ABA to the same extent, thus leading us to consider Atlpp2-2 an ABA-signalling mutant. Taken together our observations show that AtLPP2 is a part of ABA signalling and participate to the regulation of stomatal movements.

Diacylglycerol kinases activate tobacco NADPH oxidase-dependent oxidative burst in response to cryptogein

Cryptogein is a 10 kDa protein secreted by the oomycete Phytophthora cryptogea that activates defence mechanisms in tobacco plants. Among early signalling events triggered by this microbial-associated molecular pattern is a transient apoplastic oxidative burst which is dependent on the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity of the RESPIRATORY BURST OXIDASE HOMOLOG isoform D (RBOHD). Using radioactive [33 P]-orthophosphate labelling of tobacco Bright Yellow-2 suspension cells, we here provide in vivo evidence for a rapid accumulation of phosphatidic acid (PA) in response to cryptogein because of the coordinated onset of phosphoinositide-dependent phospholipase C and diacylglycerol kinase (DGK) activities. Both enzyme specific inhibitors and silencing of the phylogenetic cluster III of the tobacco DGK family were found to reduce PA production upon elicitation and to strongly decrease the RBOHD-mediated oxidative burst. Therefore, it appears that PA originating from DGK controls NADPH-oxidase activity. Amongst cluster III DGKs, the expression of DGK5-like was up-regulated in response to cryptogein. Besides DGK5-like is likely to be the main cluster III DGK isoform silenced in one of our mutant lines, making it a strong candidate for the observed response to cryptogein. The relevance of these results is discussed with regard to early signalling lipid-mediated events in plant immunity.